Secreted glycoproteins of the Wnt family act through an evolutionarily conserved signaling cascade which promotes the nuclear accumulation of beta-catenin, which interacts with members of the TCF family of DNA-binding proteins to activate target gene transcription. TCFs binds to specific sequences, but the consensus is so loose that potential TCF binding sites can be found throughout the genome. Despite this, we find that TCF is bound to specific locations in Wnt targets, corresponding to Wnt response elements (WREs). Systematic mutagenesis of a WRE revealed the presence of additional motifs that act with TCF binding sites to mediate activation by Wnt signaling. These motifs (called Helper sites) are found in several other WREs, and genome-wide searches for conserved TCF/Helper site clusters have identified other putative WREs. The functional significance of the Helper sites in these elements will be tested. The molecular mechanism of how Helper sites interact with TCF binding sites will be explored, and the trans-acting factor(s) that bind to it will be characterized. Binding of beta-catenin to TCF converts it from a transcriptional repressor to an activator. We have found that Wnt signaling promotes histone acetylation throughout target loci, which is correlated with activation of transcription. This widespread modification appears to be required to antagonize the action of factors that silence Wnt targets. The mechanisms and relationship between these processes will be explored in detail. Our data indicates that the transcriptional switch at Wnt targets involves changes in chromatin structure far beyond what was previously recognized. PROJECT NARRATIVE: The Wnt signaling pathway plays important roles in cell fate decisions during development, and is required for the maintenance of stem cell populations in adult tissues. Misregulation of the pathway plays a causal role in many human cancers. Our studies to understand the role of motifs besides TCF sites that contribute to WRE function will lead to better bioinformatic methods to identify Wnt targets in many important biological contexts. Our work on the role of chromatin modifications in regulating the TCF transcriptional switch will serve as a paradigm to study these processes in mammalian systems.